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Biological and Environmental Phenomena at the Interface
The Structure of Lung-mimetic Multilamellar Bodies with Lipid Compositions Relevant in Pneumonia Dylan Steer, Sherry Leung, Hannah Meiselman, Daniel Topgaard, and Cecilia Leal Langmuir, Just Accepted Manuscript • DOI: 10.1021/acs.langmuir.8b01359 • Publication Date (Web): 30 May 2018 Downloaded from http://pubs.acs.org on May 30, 2018
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a)
3:1 DPPC:DOPG
Intensity [arb. u.]
Low CL High CL
b)
4:1 DPPC:DOPG Low CL High CL
"diseased"
"diseased"
"healthy"
"healthy"
0.0 0.1 0.2 0.3 0.4 0.0 0.1 0.2 0.3 0.4 q [Å-1] q [Å-1] 12 9 6 3 0
mol% CL
c)
6
8 10 12 14 q [10-2 Å-1]
6
8 10 12 14 q [10-2 Å-1]
Intensity 100
d)
102
101
170
d-spacing [Å]
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60
Langmuir
3:1 4:1
150
DPPC:DOPG one phase two phase
130 110
e)
90
0 3 6 9 12 Cardiolipin (CL) [mol %]
Type I
Type II or
f) height 1
height 2
5µm
Low mobility
High mobility
Mixed Lipids
FIGURE 1 FIGURE 1 ACS Paragon Plus Environment
Langmuir
b)
High Ca2+
Low CL 0.08
0.10 0.12 q [Å-1]
High Ca2+
0.14
c) 140
Low Ca2+
130
Low CL
d-spacing [Å]
Intensity [arb. u.]
a) Low Ca2+ Intensity [arb. u.]
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60
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120 110 100
High CL 0.08
0.10 0.12 q [Å-1]
0.14
FIGURE 2
ACS Paragon Plus Environment
90
High CL 0
3
6 9 [Ca ] [mM] 2+
12
a)
Low Ca2+. 4:1 DPPC: DOPG raw data liquid-like gel-like full fit
Intensity [Arb. Units]
1.0
b)
Intensity [Arb. Units]
1.2
1.4
1.8
2.0
High Ca2+ Low Ca2+
1.2
1.4
1.6
q [Å-1] High CL. 4:1 DPPC: DOPG
1.8
2.0
High Ca2+ Low Ca2+
1.0
1.2
1.4
d) 3:1 DPPC:DOPG
1.3
1.6
q [Å-1]
Low CL. 4:1 DPPC: DOPG
1.0
c)
High CL Low CL
Intensity [arb. u.]
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60
Langmuir
Intensity [Arb. Units]
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1.6
q [Å-1]
CL
Low Ca2+
Low Ca2+ 1.5 1.6 q [Å-1]
2.0
e) 4:1 DPPC:DOPG
CL
1.4
1.8
1.3
1.4
1.5 1.6 1.7 q [Å-1]
FIGURE 3
ACS Paragon Plus Environment
Langmuir 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60
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a) unsaturated g β g2 carbons
130
g3
gα β
70
g1 gγ
65 13
b) ω1
ω2
c3
ω3
g2 O g g3
O
O
1
O
ω1 ω3
c10 c9
ω2
c3
O
c2
O
O
g1 O
g3
g2 O
β
O P O O-
O P O-
γ
60 55 C chemical shift [ppm]
DPPC
O
c4 c2
α
DP CP INEPT
α
+ N
γ
gα gγ O gβ OH
γ γ
CH2 groups c2
35
ω2 ω1 ω 3
DOPG
OH
FIGURE 4
ACS Paragon Plus Environment
c13c12 c10 c9
c8/11 (DO) c3
ω3
30
c3
ω2
25
Cardiolipin (CL) O
c2
O O
g1 g2 O
O
g3 gO αc gβcOH O
O O
O O P O
O P O O
O
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a) Low Ca2+. 3:1 DPPC: DOPG ω3 ( )
CH2 ( )
CL 15
CL 15
CL 0 72
71 70 65 δ13C [ppm]
64 63 δ13C [ppm]
62
c) Low CL. 3:1 DPPC: DOPG g2 g 3( ) g 1 ( )
CL 0 33
32 31 δ13C [ppm]
ω3 ( )
CH2 ( )
62
e) High CL. 3:1 DPPC: DOPG g2 g 3( ) g 1 ( )
Ca 12 Ca 6 Ca 0 33
32 31 δ13C [ppm]
ω3 ( )
δ13C [ppm]
64 δ13C [ppm]
63
62
f)
Ca 6
Ca 0 70 65
0.0 0.5 0
3
Ca 0 33
32
31
6 9 CL [mol%]
12
30
δ13C [ppm]
FIGURE 5
ACS Paragon Plus Environment
Low CL
0.5 0.0 0.5 fast
Ca 12
Ca 6
71
30
CH2 ( )
Ca 12
72
0.5
slow
Ca 0 64 63 δ13C [ppm]
Low Ca2+
d)
Ca 6
71 70 65 δ13C [ppm]
slow
fast
30
Ca 12
72
|CP/INEPT| signal
g 3( ) g 1 ( )
|CP/INEPT| signal
g2
b)
0
3
slow
|CP/INEPT| signal
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60
Langmuir
6 9 Ca2+ [mM]
12
High CL
0.5 0.0 0.5 fast
0
3
6 9 Ca2+ [mM]
12
Langmuir 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60
a) i)
ii) air
hypophase
air-water interface iii)
aveoli cells
Lung lipid multilamellar vesicles
b) CL
H2O
d
d
c) ii
iii
i
iv
R e p e at
FIGURE 6
ACS Paragon Plus Environment
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Langmuir
1
The Structure of Lung-mimetic Multilamellar Bodies with Lipid
2
Compositions Relevant in Pneumonia
3
Dylan Steera, Sherry Leunga, Hannah Meiselmana, Daniel Topgaardc, Cecilia
4
Leala,b,*
5
a
Department of Materials Science and Engineering, bFrederick Seitz Materials Research Laboratory. University of Illinois at Urbana-Champaign, Urbana, IL 61801
6 7
c
Division of Physical Chemistry, Center of Chemistry and Chemical Engineering, Lund
8
University. SE-221 00 Lund, Sweden
9
*Email:
[email protected] 10
Keywords: multilamellar bodies, lung lipid, solid-state NMR, SAXS, cardiolipin
11
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12
Abstract
13
The hierarchical assembly of lipids, as modulated by composition and environment, plays a
14
significant role in the function of biological membranes and a myriad of diseases. Elevated
15
concentrations of calcium ions and cardiolipin, an anionic tetra-alkyl lipid found in mitochondria
16
and some bacterial cell membranes, have been implicated in pneumonia recently. However, their
17
impact on the physicochemical properties of lipid assemblies in lungs and how it impairs alveoli
18
function is still unknown. We use Small- and Wide- Angle X-ray Scattering (S/WAXS) and
19
Solid-State Nuclear Magnetic Resonance (ssNMR) to probe the structure and dynamics of lung-
20
mimetic multilamellar bodies (MLBs) in the presence of Ca2+ and cardiolipin. We conjecture that
21
cardiolipin overexpressed in the hypophase of alveoli strongly affects the structure of lung-lipid
22
bilayers and their stacking in the MLBs. Specifically, S/WAXS data revealed that cardiolipin
23
induces significant shrinkage of the water-layer separating the concentric bilayers in
24
multilamellar aggregates. ssNMR measurements indicate that this inter-bilayer tightening is due
25
to undulation repulsion damping as cardiolipin renders the glycerol backbone of the membranes
26
significantly more static. In addition to MLB dehydration, cardiolipin promotes intra-bilayer
27
phase separation into saturated-rich and unsaturated-rich lipid domains that couple across
28
multiple layers. Expectedly, addition of Ca2+ screens the electrostatic repulsion between
29
negatively charged lung membranes. However, when cardiolipin is present, addition of Ca2+
30
results in an apparent inter-bilayer expansion likely due to local structural defects. Combining
31
S/WAXS and ssNMR on systems with compositions pertinent to healthy and unhealthy lung
32
membranes, we propose how alteration of the physiochemical properties of multilamellar bodies
33
can critically impact the breathing cycle.
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Langmuir
35
Introduction
36
Lungs critically rely on a hydrated lung lipid (often referred to as “surfactant”) layer (LS) present
37
at the interface between the pulmonary cells and the air to allow sufficient gas exchange, to
38
protect cells from contaminants and dehydration, and to modulate the surface energy of this
39
interface. The LS is composed of approximately 90% lipids and 10% proteins and coats the
40
interior of the alveolar sacks used in gas exchange with the blood stream.1, 2 In the process of
41
breathing-out the alveoli collapse and consequently shrink the exposed area of the mucus LS
42
coating. To allow re-inflation of the alveoli the LS surface must be expanded again, which
43
requires energy input postulated to be proportional to the surface tension of the coating.2 It is
44
generally accepted that perturbations of the LS coating lead to a number of health conditions due
45
to an unbalanced surface tension in alveoli.2, 3, 4, 5
46
The current understanding of LS structure and function has been acquired for decades and the
47
majority of studies model LS as a molecular interface between water and air.1, 4, 6, 7, 8 However,
48
LS is a sub-micron thick layer known to self-assemble into complex and heterogeneous 3D
49
structures.2,
50
hydrophobic tails facing towards the air and the hydrophilic headgroups immersed in the
51
hydrated hypophase. In the hypophase there is a mixture of structures including multilamellar
52
bodies –concentric shells of lipid bilayers aligned like onion layers – and tubular myelin –
53
crossed three dimensionally networked patterns of lipid bilayers.2 The role of this complex phase
54
behavior in LS functionality is often overlooked but it is expected to critically impact LS
55
performance in healthy as well as in diseased states.11
56
While the specific mechanism of action for LS continues to be an active area of research, studies
57
focused on thin lipid monolayers reveal that during lung compression the increased lateral
4, 9, 10
At the surface of the LS film there is a monolayer of lipids aligned with
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58
pressure selectively concentrates dipalmitoyl phosphatidylcholine (DPPC) lipids at the air-water
59
interface as a way to minimize surface energy.1,
60
having fully saturated chains with sixteen carbons and at physiologic temperatures packs very
61
densely with medium length scale ordering of parallel alkyl tails.13 With inhalation, the
62
multilamellar bodies (MLBs) resupply the interface with unsaturated lipids as the surface area
63
increases in order to increase the fluidity and allow respreading of the film over the alveolar
64
walls2, 14. Several studies investigate the importance of lipid composition, dynamics, packing,
65
and structure on the LS-air interface, however the structure of the hypophase is relatively
66
unexplored, despite its importance.1,
67
composition need to be considered for tubular myelin and multilamellar bodies extending beyond
68
minimal air/liquid interface models.
69
Pneumonia is the inflammation of pulmonary tissue due to infection that can lead to severe
70
breathing complications in terrestrial and marine mammals. Particularly, pneumonia is the
71
leading cause of morbidity in dolphin populations.17 While pneumonia is a well-studied
72
pathology, there is currently an incomplete understanding of the role of lung lipids in this
73
diseased state. It has been observed recently that addition of heart-derived cardiolipin and an
74
increase in Ca2+ in healthy animal model lungs are intimately related to appearance of
75
pneumonia-like symptoms.18,
76
pulmonary illness were observed to present abnormally high cardiolipin levels in lung lipid
77
extracts (10-15 mol% of total lipids) compared to healthy mice (
